KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Monoclonal ANTI-FLAG® M2-Alkaline Phosphatase antibody produced in mouse | Sigma-Aldrich | Cat#A9469 |
| Bacterial and virus strains | ||
| XL10-Gold ultracompetent cells | Agilent | Cat#200314 |
| BL21 (DE3) cells | Agilent | Cat#200132 |
| BL21(DE3)-RIL cells | Agilent | Cat#230245 |
| Chemicals, peptides, and recombinant proteins | ||
| n-Dodecyl-b-D-Maltopyranoside (DDM) | Anatrace | Cat#D310 |
| Cholesteryl hemisuccinate (CHS) | Sigma-Aldrich | Cat#C6512 |
| Adenosine 5’-triphosphate disodium salt hydrate (ATP) | Sigma-Aldrich | Cat#34369078 |
| 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385) | Tocris | Cat#1036 |
| 5’-N-Ethylcarboxamidoadenosine (NECA) | Tocris | Cat#1691 |
| Theophylline | Sigma-Aldrich | Cat#T1633 |
| [3H]4-[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol ([3H]ZM241385) | American Radiolabeled Chemicals | Cat#ART0884-50 |
| TALON Metal Affinity Resin | Gold bio | Cat#H-310-5 |
| Ni-NTA Metal Affinity Resin | Sigma-Aldrich | Product#70666 |
| 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) | Avanti | SKU#850457 |
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS) | Avanti | SKU#840034 |
| 1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’- rac-glycerol) (sodium salt) (POPG) | Avanti | SKU#840457 |
| Cholic Acid, Sodium Salt | Anatrace | Cat#S1010S |
| Isopropyl β- d-1-thiogalactopyranoside (IPTG) | Goldbio | Cat#I2481C |
| Triton X-100 | Sigma-Aldrich | Cat#T8787 |
| SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm | ThermoFischer Scientific | Cat#68100 |
| Bio-beads SM-2 Adsorbents | BioRad | Cat#1523920 |
| N-(2-Hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) | Sigma-Aldrich | Cas#7365-45-9 |
| TRIS Hydrochloride (Tris-HCl) | Sigma-Aldrich | Cas#1185-53-1 |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | Cas#60-00-4 |
| Cholesterol | Sigma-Aldrich | Cas#57-88-5 |
| Ergosterol | Cayman Chemicals | Cas#57-87-4 |
| Epi-cholesteryl methyl ether | Sigma-Aldrich | Cas#2867-93-8 |
| Digoxigenin | Sigma-Aldrich | Cas#1672-46-4 |
| Biotin | Sigma-Aldrich | Cas#58-85-5 |
| Imidazole | Sigma-Aldrich | Cas#288-32-4 |
| Glycerol | Sigma-Aldrich | Cas#56-81-5 |
| Sodium phosphate monobasic anhydrous | Sigma-Aldrich | Cas#7558-80-7 |
| Sodium phosphate dibasic anhydrous | Sigma-Aldrich | Cas#7558-79-4 |
| Magnesium Chloride | Sigma-Aldrich | Cas#7791-18-6 |
| Methanol | Sigma-Aldrich | Cas#67-56-1 |
| DMSO | Fisher Scientific | Cas#67-68-5 |
| Sodium Chloride | Sigma-Aldrich | Cas#7647-14-5 |
| Potassium Chloride | Sigma-Aldrich | Cas#7447-40-7 |
| Aldrithiol-4 | Sigma-Aldrich | Cas#2645-22-9 |
| 2,2,2-Trifluoroethanethiol (TET) | Sigma-Aldrich | Cas#1544-53-2 |
| Trifluoroacetic acid (TFA) | Sigma-Aldrich | Cas#76051 |
| N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) | Invitrogen | Cat#D346 |
| Guanosine-5’-diphosphate (disodium salt) (GDP) | ThermoFisher Scientific | Cas#7415-69-2 |
| 2-Iodoacetamide | TCI Chemicals | Cas#144-48-9 |
| Laurdan | Tocris | Cas#74515-25-6 |
| Deposited data | ||
| A2AAR in complex with ZM241385 | Jaakola et al., 200846 | PDB: 3EML |
| A2AAR in complex with NECA bound to an engineered G protein | Carpenter et al., 201644 | PDB:5G53 |
| Experimental models: Organisms/strains | ||
| P. pastoris: Bg12 | BioGrammatics | Cat#PS004-01 |
| Oligonucleotides | ||
| A2AAR[A289C,W129I] Forward: [5’AAGGGCATCATTGCCATCTGCATTGTGCTGTCGIIIGCCATCGGCCTGAC3’] | This study | n/a |
| A2AAR[A289C,W129I] Reverse: [5’GTCAGGCCGATGGCAAACGACAGCACAATGCAGATGGCAATGATGCCCTT3’] | This study | n/a |
| Recombinant DNA | ||
| Plasmid: human A2AAR (1-316) in pPIC9K | Eddy et al., 201850 | n/a |
| Plasmid: human A2AAR[A289C] | Thakur et al., 202364 | n/a |
| Plasmid: human A2AAR[A289C,R291Q] | Thakur et al., 202364 | n/a |
| Software and algorithms | ||
| GraphPad prism | GraphPad Software Inc. | https://www.graphpad.com |
| MestreNova 14.1.1-24571 | MestreLab Research S.L | https://mestrelab.com/download/mnova/ |
| Topspin 4.0.8 | Bruker Biospin | https://www.bruker.com/en/products-and-solutions/mr/nmr-software/topspin.html |
| UCSF Chimera | UCSF | https://www.cgLucsf.edu/chimera/ |
| Other | ||
| PD-10 column | Cytiva | Cat#17085101 |
| Vivaspin® 6 ultrafiltration spin columns MWCO 30 kDa | Cytiva | Cat#28932361 |
| Vivaspin® 6 ultrafiltration spin columns MWCO 10 kDa | Sigma-Aldrich | SKU#GE28932296 |
| AccuPrime Pfx SuperMix | ThermoFischer Scientific | Cat#12344040 |
| SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm | ThermoFischer Scientific | Cat#68100 |
| Amplex Red Cholesterol Assay Kit | ThermoFischer Scientific | Cat#A12216 |
| GTPase-Glo Assay | Promega | Cat#V7681 |